CN108333372A - A kind of cardic fatty acid binding protein H-FABP efficient detection test paper - Google Patents
A kind of cardic fatty acid binding protein H-FABP efficient detection test paper Download PDFInfo
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- CN108333372A CN108333372A CN201810226015.8A CN201810226015A CN108333372A CN 108333372 A CN108333372 A CN 108333372A CN 201810226015 A CN201810226015 A CN 201810226015A CN 108333372 A CN108333372 A CN 108333372A
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- antibody
- gold
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
Abstract
The invention discloses a kind of cardic fatty acid binding protein H FABP efficient detection test paper, including are located at the sample pad being sequentially connected in fixed plate, gold-labelled pad, nitrocellulose filter and absorption pad；Detection line antibody and nature controlling line antibody are equipped on nitrocellulose filter；Gold labeling antibody is equipped in gold-labelled pad, the sample pad is pad made of glass fibre cotton, the gold-labelled pad is pad made of carboxylated cellulose film, the gold labeling antibody is colour developing antibody, detection line antibody is to capture antibody, colour developing antibody can mutually match to form sandwich anti-human H FABP monoclonal antibodies with antibody is captured for 2 plants, and the gold labeling antibody is marked with 40nm nanometers of golden shells, and the gold-labelled pad is equipped with functional layer.The present invention passes through the special gold-labelled pad containing particular functional layer so that gold mark rate of release slows down, and is buffered, to make analyte and the concentration proportion of gold labeling antibody in sample increase, to improve the sensitivity of ELISA test strip.
Description
Technical field
The invention belongs to biological technology applications, and in particular to a kind of cardic fatty acid binding protein H-FABP
(Heart-type Fatty Acid Binding Protein, H-FABP) efficient detection test paper.
Background technology
H-FABP is present in myocardial cell cytoplasm, rich content.Myocardial damage early stage, cardiac muscle cell's film integrality are broken
Bad, H-FABP enters blood.H-FABP 1-2h after myocardial damage can be detected in blood.Since H-FABP cardiac muscles are special
Property higher than current clinically used myoglobins, therefore H-FABP can become a kind of important detection Acute myocardial tissue damage
Earlier specificity marker.Currently, the detection of biochemical marker is widely used to acute myocardial infarction AMI, the instability mode heart twists
Bitterly, the diagnosis of the myocardial injury diseases such as acute heart failure, myocarditis, openheart surgery lesion assessment, Newborns with Asphyxia myocardial damage, danger
Dangerous degree layering and prognosis are judged.The U.S. every year because suspect property coronary syndrome emergency treatment medical treatment patient up to 80,000,000, wherein
45% or so is related to heart, and 15% is diagnosed as acute coronary syndrome.The incidence of China's acute coronary syndrome is about
50/100000, the number for dying of acute myocardial infarction AMI every year is more than 1,000,000.Therefore, the application of H-FABP detections has important society
Meeting and economic benefit.
In notification number is CN102298055B files, when detecting sample, the adhesive sticker on test strips surface is because of blade two
The stress on side is different, causes to try the flow velocity difference that gold-labelled pad causes sample in gold-labelled pad because of thickness difference, cause with gold
Target antibody is combined uneven with the analyte in sample, eventually results in the display-detection line or control line of testing result
Broken string is generated, i.e., lines show incomplete phenomenon.In addition, sample addition concentrates additive amount to concentrate in sample, flow rate of liquid is fast,
Sample being flushed in gold-labelled pad quickly, that is, have a large amount of marker to be brought to gold-labelled pad, make the detection of gold-labelled pad sensitive instead
Degree reduces.
Invention content
Goal of the invention：For the deficiencies in the prior art, the object of the present invention is to provide a kind of cardioid aliphatic acid knots
Hop protein H-FABP efficient detection test paper, make it have easy to operate, sensitivity ＜ 3ng/mL, result stablize the advantages that.
Invention content：The present invention provides a kind of cardic fatty acid binding protein H-FABP efficient detection test paper, including set
Sample pad, gold-labelled pad, nitrocellulose filter and the absorption pad being sequentially connected in fixed plate；Inspection is equipped on nitrocellulose filter
Survey line antibody and nature controlling line antibody；Gold labeling antibody is equipped in gold-labelled pad, the sample pad is pad made of glass fibre cotton,
The gold-labelled pad is pad made of carboxylated cellulose film, and the gold labeling antibody is colour developing antibody, and detection line antibody is to capture
Antibody, colour developing antibody can mutually match to form sandwich anti-human H-FABP monoclonal antibodies, the gold with antibody is captured for 2 plants
Labeling antibody is marked with 40nm nanometers of golden shells, and the sample pad is equipped with functional layer.
Further, the functional layer based on mass fraction by 18% pure silver nitrate, 70% gelatin, 8% department's sheet 80 with
And 4% cyclodextrin forms.
The preparation method of above-mentioned cardic fatty acid binding protein H-FABP efficient detection test paper, includes the following steps：
(1) monodispersed silver nanoparticle is prepared using trisodium citrate as stabilizer and reducing agent using " seed " growth method
Particle is as template is sacrificed, by the 40nm gold nanoshells for replacing reaction acquisition colloid, high dispersive；It is as follows：With
Trisodium citrate is stabilizer and reducing agent, prepares monodispersed silver nano-grain and is used as " seed "：It will under 70 DEG C of water-bath
80mL deionized waters are heated to constant temperature, and the citric acid three sodium solution that 20mL weight percent is 1% is added, stirs evenly, is added
The AgNO3 solution of 1.7mL10mg/mL is vigorously stirred lower addition 2mL1%NaHB4 solution, then continuous heating and stirring 1h, room
It is settled to 100mL after temperature is cooling；" seed " of silver nano-grain is grown：2mL1% citric acids three are added into 80mL deionized waters
Sodium solution is simultaneously heated to fluidized state, and the silver-colored seed solution of certain volume is then added, and in being vigorously stirred process, is added
The AgNO3 solution of 1.7mL10mg/mL continues to keep stirring and heat 30min, and constant volume is to 100mL after room temperature cooling；It is received with silver
Rice grain is to sacrifice template, and colloid, high monodispersed gold nanoshell are prepared by replacing reaction：Take 30mL silver nano-grains molten
Liquid centrifuges 20min at 12500g, supernatant is poured out, then with deionized water constant volume to 100mL, by the silver nanoparticle of this 100mL
Grain solution is heated to fluidized state, and 2mM HAuCl4 solution 2mL are added with vigorous stirring, and reaction solution color is gradually turned by yellow
Become blackish green, room temperature cooling；
(2) nanogold shell solution pH8.0~8.5 are adjusted, colour developing antibody is added and is reacted with nanogold shell solution, centrifuges heavy
It forms sediment, precipitation is resuspended, obtains gold labeling antibody solution；
(3) after carboxylated cellulose film being pre-processed, drying, by that will be matched respectively by infrared spraying equipment in vacuum environment
18% pure silver nitrate, 70% macromolecule dispersant, 8% department's sheet 80 and 4% cyclodextrin made is sprayed into carboxylation fiber
Plain film forms well gold-labelled pad, then uses gold labeling antibody solution spray printing；Specific infrared spraying method can be compared in through the invention
Good is embedded in component in gold-labelled pad without influencing its performance.
(4) it is sprayed on having activated nitrocellulose filter with capture antibody and draws a detection line, vacuum drying；Then sheep anti mouse is used
IgG sprays on having activated nitrocellulose membrane draws nature controlling line, vacuum drying；
(5) it is pasted onto on the plastic plate of single side pressure sensitive adhesive, cuts by sample pad, gold-labelled pad, nitrocellulose membrane, absorption pad sequence
It cuts, is packed into plastic clip, packaging；Wherein, colour developing antibody is 2 plants of anti-human H-FABP that can form sandwich pairing mutually with antibody is captured
Antibody.
The application of above-mentioned H-FABP nanometers of gold mark detection test paper item H-FABP in detecting human blood.
Advantageous effect：The cardic fatty acid binding protein H-FABP efficient detection test paper of the present invention, uses hollow Jenner
Rice shell replaces the common spherical nanoparticle of current colloidal gold strip.It is detected applied to people H-FABP, specificity is high, due in
The color (blackish green) that empty gold nanoshell is presented can be generated under based on white background than spherical gold nano grain (kermesinus)
Preferably differentiate optical signal.
The present invention passes through the special gold-labelled pad containing particular functional layer so that and gold mark rate of release slows down, and is buffered,
To make analyte and the concentration proportion of gold labeling antibody in sample increase, to improve the sensitivity of ELISA test strip.
In addition, by the gold-labelled pad of particular functional layer, by the gelatin of special ratios and its pure silver nitrate, department this 80 and
The effect of cyclodextrin avoids the flow velocity difference that gold-labelled pad causes sample in gold-labelled pad because of thickness difference, causes with gold
Target antibody is combined uneven with the analyte in sample, eventually results in the display-detection line or control line of testing result
Broken string is generated, i.e., lines show incomplete phenomenon.
Description of the drawings
Fig. 1 is the structural schematic diagram of test strips.
Specific implementation mode
With reference to specific embodiment, the present invention is described further.
Prepared by following example is cardic fatty acid binding protein H-FABP efficient detection test paper, including is located at fixation
Sample pad 1, gold-labelled pad 2, nitrocellulose filter 3 and the absorption pad 4 being sequentially connected on plate；Inspection is equipped on nitrocellulose filter 3
Survey line antibody 5 and nature controlling line antibody 6；Gold labeling antibody is equipped in gold-labelled pad 2, the sample pad 1 is made of glass fibre cotton
Pad, the gold-labelled pad 2 be carboxylated cellulose film made of pad, the gold labeling antibody be colour developing antibody, detection line antibody
To capture antibody, colour developing antibody can mutually match to form sandwich anti-human H-FABP monoclonal antibodies, institute with antibody is captured for 2 plants
The gold labeling antibody stated is marked with 40nm nanometers of golden shells, and the gold-labelled pad is equipped with functional layer；The functional layer is based on mass fraction
It is made of 18% pure silver nitrate, 70% gelatin, 8% department's sheet 80 and 4% cyclodextrin.
(1) preparation of nanogold shell solution：
Using trisodium citrate as stabilizer and reducing agent, prepares monodispersed silver nano-grain and be used as " seed "：At 70 DEG C
Water-bath under 80mL deionized waters are heated to constant temperature, the citric acid three sodium solution of 20mL 1% (wt) is added, stirs evenly, adds
The AgNO3 solution for entering 1.7mL 10mg/mL is vigorously stirred lower addition 2mL1%NaHB4 solution, then continuous heating and stirring
1h is settled to 100mL after room temperature cooling.
" seed " of silver nano-grain is grown：1% sodium citrate solutions of 2mL are added into 80mL deionized waters and heat
To fluidized state, the silver-colored seed solution of certain volume is then added, in being vigorously stirred process, is added 1.7mL 10mg/mL's
AgNO3 solution continues to keep stirring and heat 30min, and constant volume is to 100mL after room temperature cooling.
It is to sacrifice template with silver nano-grain, colloid, high monodispersed gold nanoshell is prepared by replacing reaction：It takes
30mL silver nanoparticle solutions centrifuge 20min at 12500g, pour out supernatant, then with deionized water constant volume to 100mL.It will
The silver nanoparticle solution of this 100mL is heated to fluidized state, and 2mM HAuCl are added with vigorous stirring4Solution 2mL, reaction
Liquid color is gradually changed into blackish green, room temperature cooling by yellow.
(2) the gold label of colour developing antibody：
Nanogold shell solution pH8.0~8.5 are adjusted, colour developing antibody is added and is reacted with nanogold shell solution, centrifuges to obtain precipitation,
Precipitation is resuspended, obtains gold labeling antibody solution.
(3) prepared by gold-labelled pad and spray is padded：
After carboxylated cellulose film is pre-processed, drying, by that will be prepared respectively by infrared spraying equipment in vacuum environment
18% pure silver nitrate, 70% macromolecule dispersant, 8% department's sheet 80 and 4% cyclodextrin got well are sprayed into carboxylated cellulose
Film forms well gold-labelled pad, then uses gold labeling antibody solution spray printing.
(4) prepared by detection line：
1 plant of anti-human H-FABP monoclonal antibodies BT01 is taken to do detection line antibody, with buffer solution (10% trehalose, 6% first
Alcohol) it is diluted to 1mg/mL；Antibody is drawn on nitrocellulose filter with film instrument is drawn, a concentration of 1ul/cm forms detection line, vacuum
It is dry.
(5) prepared by nature controlling line：
Sheep anti-mouse igg is diluted to 0.5mg/mL with buffer solution (10% trehalose, 6% methanol)；Antibody is drawn with film instrument is drawn
On nitrocellulose filter, a concentration of 0.5ul/cm forms nature controlling line, vacuum drying.
(6) test strips are assembled：
Sample pad 1, gold-labelled pad 2, nitrocellulose filter 3 and blotting paper 4 are pasted onto to the plastics of single side pressure sensitive adhesive in order
On plate, as shown in Figure 1, being equipped with detection line antibody 5 and nature controlling line antibody 6 on nitrocellulose filter 3；6cm width is cut to paste
Plastic plate afterwards is packed into detection card；It is packed with the heat-sealing Fresco Bag with drier, room temperature preservation.
(7) it detects：
10000 μ l of sample are divided into 100 groups and are added dropwise to well respectively, when 15min is observed, interpretation ELISA test strip
As a result standard is：The positive has colour developing band at detection line and at nature controlling line；Feminine gender, without colour developing band, Quality Control at detection line
There is colour developing band at line；And developing sensitivity ＜ 3ng/mL, no broken string phenomenon.
The method of reference standard sample detection is detected 300 physical examination human normal plasma's samples, wherein 299
Detection line does not occur blackish green colo(u)r streak, and developing sensitivity ＜ 3ng/mL, no broken string phenomenon in 30min.
(1) purifying of Autopsy Cases H-FABP：
People's left compartment muscle is taken to be homogenized in buffer solution, with (NH after multi-step centrifugation4)2SO4Saltout → centrifugation → precipitate
Buffer solution be resuspended dialyse protein crude extract → Sephadex G-75 gel filtrations → electrophoresis determines the collection of prominent protein band
Pipe number merges collection → Q Sepharose Fast Flow anion-exchange chromatographies → polyethylene glycol concentration → buffer solution and dialyses
H-FABP.Purifying product using SIGMA companies H-FABP as reference standards, with
Tricine-SDS-PAGE electroresis appraisal relative molecular mass 15KD, purity confirm up to 90% or more, and through mass spectrum.
(2) prepared by H-FABP monoclonal antibodies
Immune animal：Select Balb/c mouse of the 8-12 week old with myeloma cell with germline, the 120 μ L purified with classical approach
H-FABP (100 μ g containing protein) is mixed well with 120 μ L Freund's complete adjuvants, is injected in mouse peritoneal, every 2 weeks 100 μ g/
H-FABP only is mixed well with equivalent freund 's incomplete adjuvant, booster immunization in injection mouse peritoneal, totally 3~4 times.
It is detected using indirect elisa method and mouse tail vein blood antibody is immunized.
Cell fusion：Higher-Balb/C the mouse of titre are selected to prepare fusion, 3d reinforces again in mouse peritoneal before merging
Immune, dosage is 50 μ g H-FABP.
Take 40mL HAT culture solutions, 15mL DMEM serum-free mediums and 1mL 50%PEG (molecular weight：2000) respectively
It is placed in pre-temperature in 37 DEG C of water-baths.Prepare 1, the 500mL beakers of 37 DEG C of water of Sheng.
Take 2.4 × 107/10mL of myeloma cell, 1.8 × 108/10mL of splenocyte that sterilizing 50mL centrifuge tubes are added respectively
In, mixing, and add DMEM serum-free mediums to 40mL.
After two kinds of cell suspensions are mixed well, 1000rpm centrifuges 10min, supernatant to the greatest extent, with finger attack tube bottom,
Two kinds of cells are made to be mixed into paste.
Centrifuge tube is placed in being filled with water in beaker of 37 DEG C of pre-temperatures, the 50%PEG solution of 0.8mL pre-temperatures is drawn with suction pipe,
It is added in 1min, stands 90s.
The serum-free medium of 37 DEG C of 15mL pre-temperatures is added dropwise immediately, PEG is made to dilute and fail.Before dropwise addition method is
30s adds 1mL；30s adds 3mL afterwards；Then 15mL is added in 3min.
DMEM serum-free mediums are added to 40mL, 1000rpm centrifugation 10min, to the greatest extent supernatant.
Add the HAT culture solutions of 40mL fetal calf serums containing 15%-20% in the cell being collected by centrifugation.It is gently mixed with suction pipe
It is even, it is added drop-wise in the aperture of 4 piece of 96 porocyte culture plates containing feeder cells, is dripped per hole 2, set 37 DEG C, the training of 7%CO2
It supports and is cultivated in case.
The clone of hybridoma and screening：Selection culture is carried out after cell fusion in HAT culture solutions.
Culture hole supernatant specific antibody is detected using indirect ELISA.
The cloning of hybridoma：Choosing positive highest hole is subcloned colony screening cell strain, sun using limiting dilution assay
Property cell strain liquid nitrogen cryopreservation.
(3) preparation of monoclonal antibody：
Before being inoculated with hybridoma, injection 0.5mL norphytanes or atoleine in Balb/c mouse peritoneals, then every
5 × 105~106 hybridomas are injected in mouse peritoneal.Hybridoma is inoculated with after about 7~10 days, mouse web portion is apparent
Swell places a test tube with its abdomen of 75% ethanol disinfection below mouse, and the injection needle of sterilizing is pierced under mouse
Abdomen allows ascites to instill in pipe.After being spaced 2~3d, after ascites accumulates again, ascites is taken again with method, is taken 2-3 times altogether, it is usually every
Mouse can harvest 5~10mL of ascites, 4 DEG C of preservations.The ascites of collection, 1500rpm centrifuge 10min, and Aspirate supernatant is added
0.02% Sodium azide.Indirect elisa method detects ascites antibody concentration, Protein A-Sepharose column purifications.It is anti-human that mouse is made
H-FABP antibody BT02.
Preparation method of the preparation method of anti-human H-FABP monoclonal antibodies BT01 with the anti-human H-FABP antibody BT02 of mouse.Choosing
It takes to match to form sandwich anti-human H-FABP monoclonal antibodies BT01 and the anti-human H-FABP antibody BT02 of mouse carrys out use.
Animal is immunized in sheep anti-mouse igg：Adult Healthy Sheep is chosen, it is complete with the mouse IgG 2ml and 2ml Fu Shi that classical approach purifies
Full adjuvant mixes well, and injection sheep is intravenous, fully mixed every the mouse IgGs of 2 weeks 2ml/ only and equivalent freund 's incomplete adjuvant
It is even, the injection intravenous booster immunization of sheep, totally 3~4 times.Sheep blood is collected, indirect elisa method detects sheep blood antibody concentration, Protein
A-Sepharose column purifications.Sheep anti-mouse igg is made.
The above is only a preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (3)
1. a kind of cardic fatty acid binding protein H-FABP efficient detection test paper, it is characterised in that：Including be located in fixed plate according to
Secondary connected sample pad (1), gold-labelled pad (2), nitrocellulose filter (3) and absorption pad (4)；It is set on nitrocellulose filter (3)
There are detection line antibody (5) and nature controlling line antibody (6)；Gold labeling antibody is equipped in gold-labelled pad (2), the sample pad (1) is glass
Pad made of glass cellucotton, the gold-labelled pad (2) are pad made of carboxylated cellulose film, and the gold labeling antibody is that colour developing is anti-
Body, detection line antibody are to capture antibody, and colour developing antibody is 2 plants with capture antibody can mutually match to form sandwich anti-human H-FABP
Monoclonal antibody, the gold labeling antibody are marked with 40nm nanometers of golden shells, and the gold-labelled pad is equipped with functional layer.
2. a kind of cardic fatty acid binding protein H-FABP efficient detection test paper according to claim 1, it is characterised in that：
The functional layer is made of 18% pure silver nitrate, 70% gelatin, 8% department's sheet 80 and 4% cyclodextrin based on mass fraction.
3. human blood H-FABP nano-gold labeled test paper described in claim 1 H-FABP in detect human blood is answered
With.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112903989A (en) * | 2021-01-27 | 2021-06-04 | 中国人民解放军军事科学院军事医学研究院 | Heart-type fatty acid binding protein rapid detection card |
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| CN1866017A (en) * | 2005-05-16 | 2006-11-22 | 艾博（武汉）生物技术有限公司 | Three-in-one detection reagent plate for early diagnosis of acute myocardial infarction |
| CN201903547U (en) * | 2010-11-29 | 2011-07-20 | 深圳市博卡生物技术有限公司 | Rapid detection test strip |
| CN102298055A (en) * | 2011-07-21 | 2011-12-28 | 南京博天科智生物技术有限公司 | Human blood H-FABP nano-gold labeled test paper and preparation method thereof |
| CN103901216A (en) * | 2014-04-22 | 2014-07-02 | 上海科华生物工程股份有限公司 | Human H-FABP colloidal gold test paper and preparation method thereof |
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- 2018-03-19 CN CN201810226015.8A patent/CN108333372A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996024060A1 (en) * | 1995-02-03 | 1996-08-08 | British Biocell International Limited | Assay device and method |
| CN1866017A (en) * | 2005-05-16 | 2006-11-22 | 艾博（武汉）生物技术有限公司 | Three-in-one detection reagent plate for early diagnosis of acute myocardial infarction |
| CN201903547U (en) * | 2010-11-29 | 2011-07-20 | 深圳市博卡生物技术有限公司 | Rapid detection test strip |
| CN102298055A (en) * | 2011-07-21 | 2011-12-28 | 南京博天科智生物技术有限公司 | Human blood H-FABP nano-gold labeled test paper and preparation method thereof |
| CN103901216A (en) * | 2014-04-22 | 2014-07-02 | 上海科华生物工程股份有限公司 | Human H-FABP colloidal gold test paper and preparation method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112903989A (en) * | 2021-01-27 | 2021-06-04 | 中国人民解放军军事科学院军事医学研究院 | Heart-type fatty acid binding protein rapid detection card |
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Application publication date: 20180727 |